Pain is a serious, debilitating condition that is under-treated because of the dose-limiting side effects of currently used opiate drugs such as morphine. Enkephalin is an endogenous opiate peptide, which induces analgesia, but unlike morphine it has few, if any side effects. Importantly, it does not have the abuse potential of opiate alkaloids. Because it does not cross the blood brain barrier, enkephalin itself cannot be employed therapeutically. However, drugs that could increase the levels of enkephalin in the nervous system could be a new treatment for pain with major advantages over presently used narcotics. Enkephalins are produced by a series of proteolytic events that cleave the large protein precursor, proenkephalin, to the smaller biologically active enkephalin pentapeptide. We have identified the major proenkephalin cleaving activity in secretory vesicles for the generation of enkephalin, and it has been demonstrated as the cysteine protease cathepsin L. Cathepsin L cleaves at appropriate proenkephalin processing sites, is colocalized with enkephalin in secretory vesicles, and brain levels of enkephalin are reduced in cathepsin L knockout mice. These results demonstrate the importance of cathepsin L for production of active enkephalin. Notably, cathepsin L activity is tonically inhibited in vivo by the endogenous serpin protease inhibitor endopin 2. We propose that drugs can be developed to prevent endopin 2 from inhibiting cathepsin L, thereby increasing cathepsin L activity for processing proenkephalin to elevate enkephalin in the central nervous system (CNS) and enhance analgesia for pain relief. In this 6-month Phase I project, the first specific aim will develop and optimize a fluorescent-based high throughput screening (HTS) assay to measure cathepsin L activity in the presence of endopin 2, as specific aim 1. This high throughput assay will allow screening of a chemical library to identify compounds that increase cathepsin L activity, which would occur by interrupting endopin 2 modulation of cathepsin L. The second specific aim will utilize these HTS assays to screen a small molecule library for compounds that activate cathepsin L activity, in the presence of endopin 2, to increase enkephalin production. Lead hits can be developed in future studies (phase II SBIR) into novel drugs to increase enkephalin production that would provide pain relief without the side effects of presently used opiate narcotics.